The present invention relates to novel compositions which desorb bacteria and other microorganisms from solid surfaces and from living tissue.
The main use contemplated for the compositions of the present invention is in oral hygiene. Compositions of the invention can also be used in entirely different applications, where microorganisms are to be removed from surfaces to which they are attached, or where enhanced microbial adhesion to oil droplets is desired.
According to the present invention there is now provided a composition for desorbing bacteria from solid surfaces and from living tissues, which is in the form of a two-phase preparation, and which upon shaking forms a temporary oil-in-water emulsion of limited lifetime said composition comprising:
a) about 50 to about 97% w/w of an aqueous phase;
b) about 3 to about 50% w/w of a water immiscible oily phase, comprising a vegetable oil, a mineral oil, a pharmacologically acceptable aliphatic hydrocarbon or a mixture thereof; and
c) about 0.003 to about 2.0% w/w of an amphipathic cationic moiety in an effective amount to enable the formation of said temporary oil-in-water emulsion which emulsion breaks down and separates within a period of about 10 seconds to thirty minutes of the formation thereof.
Amongst edible oils, which can be used to form said water immiscible oily phase there may be mentioned oils such as olive oil, corn oil, coconut oil, soybean oil, safflower oil. There can also be used a wide variety of pharmacologically acceptable hydrocabons such as octane, decane, tetradecane, hexadecane, xylene, white mineral oil, and mixtures thereof etc.
Amongst suitable colors, there may be mentioned colors of triphenylmethane, naphthol, xanthene, monoazo, pyrazol, anthraquinone and cationic colors; examples include Food Blue 2 and its ammonium salt; DandC Yellow No. 7, DandC Yellow No. 10, DandC Yellow Nos. 4, 6, 22, 28, 33 or 40; DandC Green No. 5, DandC Orange No. 11, DandC Red Nos. 19 and 37, Basic Blue Nos. 6, 9, 41, 99, etc.
Preferably said amphipathic cationic moiety is selected from pyridinium core surface-active cationic molecules such as cetylpyridinium chloride, laurylpyridinium chloride, etc.; from chlorhexidines such as chlorhexidine, its diacetate, chlorhexidine digluconate, chlorhexidine dihydrochloride; from monalkyl quaternary ammonium compounds (quats) such as benzalkonium chloride, cetalkonium chloride and bromide, lauralkonium chloride and bromide, soytrimonium chloride, PEG-5 stearyl ammonium lactate; from dialkyl quaternary ammonium compounds (diquats) such as dilauryl dimonium chloride, dicetyl dimonium chloride and bromide, dequalinium chloride, soyamido propyl benzyldimonium chloride, quaterniums such as quaternium 15 and polyquaterniums, etc; amine fluorides, from cationic polysaccharides, such as chitosan and its derivatives; from cationic polypeptides, such as poly L-lysine, poly D-lysine, lysozyme.
In especially preferred embodiments of the present invention said amphipathic cationic moiety is selected from cetylpyridinium chloride, a chlorhexidine compound, chitosan, chitin derivatives, poly L-lysine and lysozyme.
The invention also provides a method for enhancing microbial adhesion at an oil-water interface comprising admixing an aqueous microbial suspension and oil in the presence of an amphipathic cationic moiety.
In U.S. Pat. No. 4,525,342, Weiss et al. have described an essentially detergent free mouthwash based on a combination of an oily phase and an aqueous one. This composition must be marketed in a two-compartment squirt bottle, and the user swishes the two phases in his mouth in order to attain the desired effect. The composition is based on the attachment of amphipathic substances to the oil droplets during the swishing action, thus coating the oil droplets. The presence of detergents is reported to be deleterious in said patent. Various deleterious physiological effects have been reported regarding the use of detergents in the oral cavity. It has been reported that the presence of detergents prevents, or at least decreases adhesion of microorganisms to oil droplets as is taught, e.g. by the statement: xe2x80x9cThe aqueous bacterial suspension must be free of surfactants, which inhibit the assayxe2x80x9d (M. Rosenberg, 1984. Bacterial Adherence to Hydrocarbons: A Useful Technique for Studying Cell Surface Hydrophobicity. FEMS Microbiology Letters 22, 289-295). In a recent review on hydrophobic interactions and adhesion, the authors write: xe2x80x9cOur appreciation of the extent to which hydrophobic interactions mediate various bacterial adhesion phenomena could be greatly augmented by including agents that interfere with hydrophobic interactions (e.g., surfactants, chaotrophic agents) in adhesion assays (M. Rosenberg and S. Kjellerberg, 1986, Hydrophobic Interactions: Role in Bacterial Adhesion. Advances in Microbial Ecology 9, 353-393). Moreover, in a previous study which did examine the effect of some cationic amphipathic antibacterial agents on adhesion of relatively non-hydrophobic Escherichia coli strains to xylene, the cells were exposed to the cationic agents, but the cationic agents were subsequently removed by washing the cell pellet prior to addition of the xylene, presumably to avoid inhibition of the adhesion in the presence of the cationic agents (B. M. A. El-Falana, D. T. Rogers, A. D. Russell and J. R. Furr. 1985 Effect of Some Antibacterial Agents on the Hydrophobicity of Wild-Type and Envelope Mutant of Escherichia coli. Current Microbiology 12, 187-190). Similarly, chlorhexidine, an amphipathic, surface-active cationic agent commonly incorporated into mouthwashes has been shown in many studies to inhibit microbial adhesion to surfaces. For example, J. McCourtie, T. W. MacFarlane and L. P. Samaranayake (Effect of saliva and serum on the adherence of Candida species to chlorhexidine-treated denture acrylic, J. Med. Microbiology, 21:209-213, 1986) showed that treatment of saliva- or serum-coated acrylic with chlorhexidine gluconate reduced adherence by between 19 and 86%.
Moreover, whereas salts such as sodium chloride have been generally considered to enhance microbial adhesion to surfaces, it has now been found that the presence of inorganic cations, such. as sodium and magnesium cations, inhibit the stimulation of adhesion brought about by cationic moieties such as cetylpyridinium, chlorhexidine, chitosan, poly L-lysine, poly D-lysine and lysozyme.
The main use of the compositions is in the form of mouthwashes, which effectively remove a large percentage of microorganisms, debris and other odor-causing materials from the surface of teeth and from the oral cavity. Thus especially preferred embodiments of the present invention relate to a mouthwash as defined above comprising about 0.003 to about 0.5 w/w of an amphipathic cationic moiety in an effective amount to enable the formation of said temporary oil-in-water emulsion which emulsion breaks down and separates within a period of about 10 seconds to thirty minutes of the formation thereof.
The novel compositions comprise, in combination, an organic water-immiscible phase and an aqueous phase, and a small quantity of an amphipatic cationic moiety, which is adequate to form upon vigorous mixing an emulsion of brief life-time. In addition, the composition may comprise additional cationic agent or agents which enhance adhesion to oil droplets, as shall be illustrated later. Optimally, the composition should be relatively free from interfering cations, such as sodium, potassium and magnesium and therefore preferably contain only up to 0.3% of inorganic salts.
In use, the two phases are mixed by shaking, and an emulsion is formed which is used for swishing in the mouth, and which has a limited life time, of the duration of about 10 seconds to about 30 minutes. A small amount of amine fluoride which tends to stabilize the emulsion for a brief period of time and has other beneficial (e.g. anticaries, antibacterial) effects may also be added.
There may also be incorporated certain additives such as fragrances, colors and the like.
While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.
The following experimental results illustrate the invention, both in regard to the specific formulations and also as regards the mechanism of action of the emulsions of the present invention as regards removal of dental plaque and adhering bacterial and other microorganisms.
The mouthwash compositions of the invention preferably comprise a small quantity of an amphipatic cationic moiety which is preferrably a surface-active agent which is able to form an emulsion of limited life time. The emulsion ought to be formed upon shaking of the container which contains the two separate phases, and it should preferably remain in emulsion form for at least about 20 seconds. Experiments have shown that compositions of the invention form temporary emulsions which separate after a period of about 10 seconds to 10 minutes.
We have found the unexpected result that small amounts of cationic surface-active agents promote, rather than inhibit, adhesion of bacteria and oral debris, to oil droplets. We have also found the surprising and unexpected results that sodium and magnesium chloride inhibit, rather than enhance, microbial adhesion in the presence of such cationic agents as (cetylpyridinium chloride, chlorhexidine digluconate and chitosan.
The following experiments demonstrate the ability of the cationic surfactant, cetylpyridinium chloride, to enhance microbial adhesion to oil droplets in various experiments. In one experiment Acinetobacter calcoaceticus RAG-1 (ATCC 31012) cells were grown overnight with vigorous shaking at 30 C in brain heart infusion broth. The cells were harvested by centrifugation and washed twice in 0.2% saline. The cells were then suspended in 0.2% saline to a corrected optical density of about 15 at 400 nm (1 cm light path). To 1.2 ml of suspended bacteria in 4 ml square disposable polystyrene cuvettes were added the following: 0.16 ml of 0.5% detergent in water (or water for control) and 0.24 ml water. Following brief mixing, 0.2 ml of oil was added (vs. controls with no oil added) and the mixtures vortexed vigorously for 2 min. Following about thirty minutes, the corrected turbidity of the lower aqueous phase was measured at 400 nm. Adhesion is calculated as the percent drop in turbidity following the mixing procedure. The results are summarized in the following Table.
As can be seen from the above Table, in each case, cetylpyridinium chloride greatly enhanced bacterial adhesion to the oil droplets. This phenomenon was also observed microscopically. Enhanced adhesion was not observed for non-cationic surface-active agents: adhesion to all three test oils in the presence of the anionic sodium lauryl sulphate or the nonionic Tween 20, added at the same concentration as the cetylpyridinium chloride (w/v), was 0%. Morever, the enhanced adhesion can be shown also for microbial cells other than RAG-1 which have little or no affinity for oil droplets. For example, the adhesion of Escherichia coli CSH 57 to hexadecane could be increased from 0 to 98% when 0.08% cetylpyridinium chloride was present; adhesion of the yeast Candida alpicans rose from 20 to 100% in the presence of 0.16% chlorhexidine digluconate. Adhesion of Acinetobacter calcoaceticus MR-481 rose from 0 to 97% in the presence of 0.18% of cetylpyridinium chloride.
In another experiment, a volunteer swished his mouth with 0.2% saline solution (10 ml) for thirty seconds. To 1 ml of the expectorate were ad ed 0.18 ml of 0.5% cetylpyridinium chloride (water in the case of the control) and 0.44 mm 0.2% saline. Mineral oil 50 (0.2 ml) was then added and the mixtures vortexed for 2 minutes. Following phase separation, the mixtures were examined. Whereas the turbidity of the expectorate was greatly reduced in the presence of cetylpyridinium chloride, presumably due to adsorption of bacteria and debris to the oil droplets, in the absence of this detergent, no increase in turbidity was observed.
The ability of another cationic surface-active agent, chlorhexidine digluconate, to enhance microbial adhesions to oil droplets is shown in the following experiment. Actnetobacter calcoaceticus RAG-1 (ATCC 31012) cells were grown overnight with vigorous shaking at 30 C in brain heart infusion broth. The cells were harvested, washed by centrifugation, and suspended in 0.2% saline to a corrected optidal density of ca. 18 at 0 nm (1 cm light path). To 1.2 ml of suspended bacteria in 4 ml square disposable polystyrene cuvettes were added the following: 0.16 ml of 0.5% chlorhexidine digluconate in water (or water for control) and 0.24 ml water. To each cuvette 0.2 ml of oil was added (vs. control with no oil added) and the mixtures vortexed vigorously for 2 min. After about thirty minutes, the turbidity of the lower aqueous phase was measured at 400 nm and corrected for non-linearity by a standard curve. Adhesion is calculated as the percent drop in corrected turbidity following the mixing procedure. The results are summarized in the following Table.
As can be seen from the above Table, in each case chlorhexidine digluconate greatly enhances bacterial adhesion to the oil droplets. Moreover, the enhanced adhesion can be shown also for microbial cells other than RAG-1 which have little or no affinity for oil droplets. For example, the adhesion of Escherichia coli CSH 57 to hexadecane could be increased from 0 to 98% when 0.1% chlorhexidine digluconate was present.
Adhesion to oil droplets may also be enhanced by polymeric cationic agents, such as chitosan. For example, the adhesion of Escherichia coli CSH 57 to hexadecane could be increased from 0 to 97% when 0.015% chitosan was present; adhesion of the yeast Candida albicans rose from 20 to 95% in the presence of 0.025% chitosan. Adhesion of Acinetobacter calcoaceticus MR-481 rose from 0 to 99% in the presence of 0.019% of chitosan.
The compositions of the invention are suitable for a variety of applications. As a two-phase mouthwash, containing an aqueous phase and an oil phase, the presence of adhesion-enhancing cationic agent (or combination thereof) could remove and bind high levels of oral microorganisms and debris.
The enhanced Adhesion of RAG-1 cells to oils in the presence of cationic surfactants similarly teaches the use of cationic surface-active agents to enhance microbial adhesion to oils for commercial purposes, such as immobilization of cells onto oil droplets, or of cells, cell walls, and the like for enhanced adhesion to adjuvant oils in the vaccination field.
In general, salts such as sodium chloride, magnesium chloride and various phosphates, enhance adhesion of microorganisms to surface by countering the net negative charges which generally exist on cell surfaces, and often on substrata surfaces. Weiss et al. include in their U.S. Pat. No. 4,525,342, a requirement for salt. However, as stated, it has now been found that the enhancement of microbial adhesion to oils observed in the presence of cetylpyridinium chloride, chlorhexidine gluconate and chitosan is inhibited by sodium chloride and magnesium chloride.
For example, adhesion of C. albicans to hexadecane in the presence of 0.16% cetyl pyridinium chloride fell from 100% to 18% when 0.8% NaCl was present.
In order to obtain a picture as to the efficacy of the compositions of the invention for oral use these were compared with existing mouthwashes as regards their ability to remove a given bacterial film from a solid surface.